Sylvie Jégou

Cloning, sequence analysis and tissue distribution of the mouse and rat urotensin II precursors.

Urotensin II (UII) is a cyclic neuropeptide initially isolated from the caudal neurosecretory system of teleost fish. The recent cloning of the UII precursor in frog and human has demonstrated that the peptide is not restricted to the fish urophysis but that it is also expressed in the central nervous system of tetrapods. Here, we describe the characterization of the cDNAs encoding prepro‐UII in mouse and rat. A comparison of the primary structures of mouse and rat UII with those of other vertebrate UII reveals that the sequence of the cyclic region of the molecule (CFWKYC) has been fully conserved. In contrast, the N‐terminal flanking domain of prepro‐UII has markedly diverged with only 48% sequence identity between the mouse or rat and the human precursors. In situ hybridization histochemistry showed that the prepro‐UII gene is predominantly expressed in motoneurons of the brainstem and spinal cord, suggesting that UII may play a role in the control of neuromuscular functions.

Localization and identification of Neuropeptide Y (NPY)-like immunoreactivity in the frog brain

The distribution of neuropeptide Y (NPY) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. NPY-like containing perikarya were localized in the infundibulum, mainly in the ventral and dorsal nuclei of the infundibulum, in the preoptic nucleus, in the posterocentral nucleus of the thalamus, in the anteroventral nucleus of the mesencephalic tegmentum, in the part posterior to the torus semicircularis, and in the mesencephalic cerebellar nucleus. Numerous perikarya were also distributed in all cerebral cortex. Important tracts of immunoreactive fibers were found in the infundibulum, in the preoptic area, in the lateral amygdala, in the habenular region, and in the tectum. The cerebral cortex was also densely innervated by NPY-like immunoreactive fibers. A rich network of fibers was observed in the median eminence coursing towards the pituitary stalk. Scattered fibers were found in all other parts of the brain except in the cerebellum, the nucleus isthmi and the torus semicircularis, where no immunoreactivity could be detected. NPY-immunoreactive fibers were observed at all levels of the spinal cord, with particularly distinct plexus around the ependymal canal and in the distal region of the dorsal horn. At the electron microscope level, NPY containing perikarya and fibers were visualized in the ventral nuclei of the infundibulum, using the peroxidase-antiperoxidase and the immunogold techniques. NPY-like material was stored in dense core vesicles of 100 nm in diameter. A sensitive and specific radioimmunoassay was developed. The detection limit of the assay was 20 fmole/tube. The standard curves of synthetic NPY and the dilution curves for acetic acid extracts of cerebral cortex, infundibulum, preoptic region, and mesencephalon plus thalamus were strictly parallel. The NPY concentrations measured in these regions were (pmole/mg proteins) 163 +/- 8, 233 +/- 16, 151 +/- 12 and 60 +/- 13, respectively. NPY was not detectable in cerebellar extracts. After Sephadex G-50 gel filtration of acetic acid extracts from whole frog brain, NPY-like immunoreactivity eluted in a single peak. Reverse phase high performance liquid chromatography (HPLC) and radioimmunoassay were used to characterize NPY-like peptides in the frog brain. HPLC analysis revealed that infundibulum, preoptic area and telencephalon extracts contained a major peptide bearing NPY-like immunoreactivity. The retention times of frog NPY and synthetic porcine NPY were markedly different. HPLC analysis revealed also the existence, in brain extracts, of several other minor components cross-reacting with NPY antibodies.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunohistochemical localization and radioimmunoassay of corticotropin-releasing factor in the forebrain and hypophysis of the frog Rana ridibunda

The distribution of immunoreactive corticotropin-releasing hormone (CRF) in the forebrain and pituitary of the frog Rana ridibunda was studied by means of specific radioimmunoassay and immunohistochemistry using the indirect immunofluorescence and the peroxidase-antiperoxidase techniques. Relatively high concentrations of CRF-like material were found in both chiasmatic and infundibular regions of the hypothalamus (352 +/- 11 and 422 +/- 36 pg, respectively). Large amounts of CRF were also found in neurointermediate lobe extracts. Standard curves of synthetic CRF and the dilution curves for hypothalamic or neurointermediate lobe extracts were parallel. After Sephadex G-75 gel filtration, CRF-like immunoreactivity eluted in a single peak, in the same position as synthetic ovine CRF. Reversed-phase high-performance liquid chromatography of the material purified on Sephadex G-75 revealed 5 components with CRF-like immunoreactivity. The major peak had a retention time of 22 min as compared to 25.4 min for ovine CRF and 36 min for rat CRF. The detection of CRF-like immunoreactivity in neurons was facilitated by colchicine pretreatment of the frogs. The great majority of the CRF-positive perikarya were seen in the ventral region of the preoptic nucleus. A few scattered perikarya were also observed in the dorsal preoptic nucleus and in the retrochiasmatic region. Immunoreactive fibers were found in the infundibular nucleus and in various extrahypothalamic zones. CRF-containing neurons were apparently distinct from mesotocinergic and vasotocinergic neurons. A large number of immunoreactive nerve fibers were observed in the median eminence in close contact with the capillaries of the pituitary portal plexus and in the neural lobe. A few CRF-positive fibers were detected in the intermediate lobe, whereas the distal lobe was totally negative. These results show that the diencephalon and pars intermedia-nervosa of the frog contain a peptide immunologically related to mammalian CRF.

Comparative Distribution of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Binding Sites and PACAP Receptor mRNAs in the Rat Brain During Development

The distribution and density of pituitary adenylate cyclase‐activating polypeptide (PACAP) binding sites as well as PACAP‐specific receptor 1 (PAC1‐R), vasoactive intestinal polypeptide/PACAP receptor 1 (VPAC1‐R), and VPAC2‐R mRNAs have been investigated in the rat brain from embryonic day 14 (E14) to postnatal day 8 (P8). Significant numbers of binding sites for the radioiodinated, 27‐amino‐acid form of PACAP were detected as early as E14 in the neuroepithelia of the metencephalon and the myelencephalon. From E14 to E21, the density of binding sites in the germinative areas increased by 3‐ to 5‐fold. From birth to P12, the density of binding sites gradually declined in all neuroepithelia except in the external granule cell layer of the cerebellum, where the level of binding sites remained high during the first postnatal weeks. Only low to moderate densities of PACAP binding sites were found in regions other than the germinative areas, with the exception of the internal granule cell layer of the cerebellum, which contained a high density of sites. The localization of PACAP receptor mRNAs was investigated by in situ hybridization using [35S] uridine triphosphate‐specific riboprobes. The evolution of the distribution of PAC1‐R and VPAC1‐R mRNAs was very similar to that of PACAP binding sites, the concentration of VPAC1‐R mRNA being much lower than that of PAC1‐R mRNA. In contrast, intense expression of VPAC2‐R mRNA was observed in brain regions other than germinative areas, such as the suprachiasmatic, ventral thalamic, and dorsolateral geniculate nuclei. The discrete localization of PACAP binding sites as well as PAC1‐R and VPAC1‐R mRNAs in neuroepithelia during embryonic life and postnatal development strongly suggests that PACAP, acting through PAC1‐R and/or VPAC1‐R, may play a crucial role in the regulation of neurogenesis in the rat brain. J. Comp. Neurol. 425:495–509, 2000.

Comparative effects of corticotropin-releasing factor, arginine vasopressin, and related neuropeptides on the secretion of ACTH and α-MSH by frog anterior pituitary cells and neurointermediate lobes in vitro

The ability of corticoliberin (CRF), urotensin I, sauvagine, arginine-vasopressin (AVP), and mesotocin to stimulate ACTH release by frog anterior pituitary cells and alpha-melanotropin (MSH) by frog neurointermediate lobe was studied in vitro using a perifusion technique. CRF and AVP were found to be potent stimulators of ACTH secretion, whereas urotensin I and sauvagine were totally inactive. In opposition to recent findings in the rat. CRF did not modify alpha-MSH secretion by the frog neurointermediate lobe. Mesotocin, which is present in the parenchymal cells of the frog pars intermedia, had no effect on alpha-MSH release in vitro. No potentiation of CRF-induced ACTH release was observed when anterior pituitary cells were incubated with a combination of AVP and CRF. Together with the recent elucidation of a CRF-like molecule in the frog diencephalon, these results suggest that, in Amphibia, CRF and AVP exert their stimulatory action specifically on distal lobe corticotrophs.

Melanocortin‐3 Receptor mRNA Expression in Pro‐Opiomelanocortin Neurones of the Rat Arcuate Nucleus

The melanocortins α‐ and γ‐melanocyte‐stimulating hormones (α‐ and γ‐MSH) derive from the pro‐opiomelanocortin (POMC) precursor. Melanocortins exert a wide range of biological activities in the brain through activation of at least three distinct melanocortin receptor (MC‐R) subtypes. In order to determine whether POMC neurones can modulate their own activity, we looked for the possible expression of the MC3‐R gene in POMC‐positive cell bodies in the rat hypothalamus. In situ hybridization experiments revealed that the density of MC3‐R mRNA is particularly high in the arcuate nucleus which contains the main population of POMC neurones in the brain. The occurrence of MC3‐R mRNA in POMC‐positive cell bodies was demonstrated using a double‐labelling in situ hybridization technique. The proportion of POMC neurones expressing MC3‐R mRNA was significantly higher in the most rostral (43.5%) than in the most posterior part of the arcuate nucleus (8.2%). These results indicate that melanocortins likely exert a direct regulatory feedback on POMC neurones through activation of MC3‐R receptors. Our data also suggest that MC3‐R may be involved in the neuroendocrine responses induced by centrally administered melanocortins.

In vitro study of frog (Rana ridibunda Pallas) interrenal function by use of a simplified perifusion system. II. Influence of adrenocorticotropin upon aldosterone production.

In order to investigate various factors capable of regulating frog adrenal steroidogenesis, Rana ridibunda adrenal fragments were continuously perifused for 10 hr with amphibian culture medium (ACM). Aldosterone concentrations in the effluent medium were assayed without prior extraction by means of a sensitive and highly specific radioimmunoassay method. In all the experiments, large amounts of aldosterone were secreted even in the absence of ACTH stimulation. Aldosterone output paralleled temperature variations (5 to 30°). A highly significant correlation (r = 0.982; P < 0.01) was established between the outputs of corticosterone and aldosterone during this experiment. Infusion of frog distal lobe extract gave a dose-related response, with the highest dose (0.08 distal lobe eq/ml) yielding a 6.7-fold increase in aldosterone output. In this experiment, interrenal tissue produced two times as much aldosterone as corticosterone. When various dilutions of frog intermediate lobe extracts were tested upon interrenal fragments, a linear log-dose response in aldosterone production was observed for the lower doses and a plateau was reached for the higher doses (0.05 and 0.1 intermediate lobe eq/ml). Dibutyryl cyclic AMP, at a dose of 10 mM, led to a 3.18-fold increase in aldosterone output. These results suggest that aldosterone secretion is controlled by ambient temperature and by circulating levels of adrenocorticotropin. They demonstrate that, in frogs, output of aldosterone is two times higher than output of corticosterone. The aldosterone-corticosterone ratio is even larger after stimulation by high doses of ACTH. Finally, they confirm the presence of large concentrations of biologically active corticotropin in the intermediate lobe of frog pituitary.

Localization and identification of α-melanocyte-stimulating hormone (α-MSH) in the frog brain

Abstract The distribution of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. α-MSH-like containing perikarya were localized in the infundibular region, mainly in the ventral hypothalamic nucleus. A rich plexus of immunoreactive fibers directed towards the ventral telencephalic region was detected. Reverse-phase high-performance liquid chromatography and radioimmunoassay were used to characterize α-MSH-like peptides in the frog brain. Chromatographic separation revealed that immunoreactive α-MSH coeluted with synthetic des-Nα-acetyl α-MSH, authentic α-MSH and their sulfoxide derivatives. The heterogeneity of α-MSH-like material in the frog brain was in marked contrast with the figure observed in the intermediate lobe of the pituitary gland where only des-Nα-acetyl α-MSH is present. These findings support the existence of discrete α-MSH immunoreactive neurons in the frog brain containing both desacetyl and authentic α-MSH.

Pituitary adenylate cyclase-activating polypeptide inhibits food intake in mice through activation of the hypothalamic melanocortin system.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and the proopiomelanocortin (POMC)-derived peptide, α-melanocyte-stimulating hormone (α-MSH), exert anorexigenic activities. While α-MSH is known to inhibit food intake and stimulate catabolism via activation of the central melanocortin-receptor MC4-R, little is known regarding the mechanism by which PACAP inhibits food consumption. We have recently found that, in the arcuate nucleus of the hypothalamus, a high proportion of POMC neurons express PACAP receptors. This observation led us to investigate whether PACAP may inhibit food intake through a POMC-dependent mechanism. In mice deprived of food for 18 h, intracerebroventricular administration of PACAP significantly reduced food intake after 30 min, and this effect was reversed by the PACAP antagonist PACAP6-38. In contrast, vasoactive intestinal polypeptide did not affect feeding behavior. Pretreatment with the MC3-R/MC4-R antagonist SHU9119 significantly reduced the effect of PACAP on food consumption. Central administration of PACAP induced c-Fos mRNA expression and increased the proportion of POMC neuron-expressing c-Fos mRNA in the arcuate nucleus. Furthermore, PACAP provoked an increase in POMC and MC4-R mRNA expression in the hypothalamus, while MC3-R mRNA level was not affected. POMC mRNA level in the arcuate nucleus of PACAP-specific receptor (PAC1-R) knock-out mice was reduced as compared with wild-type animals. Finally, i.c.v. injection of PACAP provoked a significant increase in plasma glucose level. Altogether, these results indicate that PACAP, acting through PAC1-R, may inhibit food intake via a melanocortin-dependent pathway. These data also suggest a central action of PACAP in the control of glucose metabolism.

Localization of the urotensin II receptor in the rat central nervous system.

The vasoactive peptide urotensin II (UII) is primarily expressed in motoneurons of the brainstem and spinal cord. Intracerebroventricular injection of UII provokes various behavioral, cardiovascular, motor, and endocrine responses in the rat, but the distribution of the UII receptor in the central nervous system (CNS) has not yet been determined. In the present study, we have investigated the localization of UII receptor (GPR14) mRNA and UII binding sites in the rat CNS. RT‐PCR analysis revealed that the highest density of GPR14 mRNA occurred in the pontine nuclei. In situ hybridization histochemistry showed that the GPR14 gene is widely expressed in the brain and spinal cord. In particular, a strong hybridization signal was observed in the olfactory system, hippocampus, olfactory and medial amygdala, hypothalamus, epithalamus, several tegmental nuclei, locus coeruleus, pontine nuclei, motor nuclei, nucleus of the solitary tract, dorsal motor nucleus of the vagus, inferior olive, cerebellum, and spinal cord. Autoradiographic labeling of brain slices with radioiodinated UII showed the presence of UII‐binding sites in the lateral septum, bed nucleus of the stria terminalis, medial amygdaloid nucleus, anteroventral thalamus, anterior pretectal nucleus, pedunculopontine tegmental nucleus, pontine nuclei, geniculate nuclei, parabigeminal nucleus, dorsal endopiriform nucleus, and cerebellar cortex. Intense expression of the GPR14 gene in some hypothalamic nuclei (supraoptic, paraventricular, ventromedian, and arcuate nuclei), in limbic structures (amygdala and hippocampus), in medullary nuclei (solitary tract, dorsal motor nucleus of the vagus), and in motor control regions (cerebral and cerebellar cortex, substantia nigra, pontine nuclei) provides the anatomical substrate for the central effects of UII on behavioral, cardiovascular, neuroendocrine, and motor functions. The occurrence of GPR14 mRNA in cranial and spinal motoneurons is consistent with the reported autocrine/paracrine action of UII on motoneurons. J. Comp. Neurol. 495:21–36, 2006.